Human growth hormone (hGH) and agonist variants thereof are members of a family of recombinant proteins, described in U.S. Pat. Nos. 4,658,021 and 5,633,352. Their recombinant production and methods of use are detailed in U.S. Pat. Nos. 4,342,832, 4,601,980; 4,898,830; 5,424,199; and 5,795,745. Human growth hormone participates in various aspects of the regulation of normal human growth and development. Through interaction with its receptors, this 22 kDa pituitary hormone modulates a multitude of biological effects, such as linear growth (somatogenesis), lactation, activation of macrophages, and insulin-like and diabetogenic effects. Chawla, Annu. Rev. Med., 34: 519 (1983); Edwards et al., Science, 239: 769 (1988); Isaksson et al., Annu. Rev. Physiol., 47: 483 (1985); Thomer and Vance, J. Clin. Invest., 82: 745 (1988); Hughes and Friesen, Annu. Rev. Physiol., 47: 469 (1985).
The administration of glycosylated and non-glycosylated peptides for engendering a particular physiological response is well known in the medicinal arts. Both purified and recombinant hGH have been used for treating conditions and diseases due to hGH deficiency, e.g., dwarfism in children. A principal factor that has limited the use of therapeutic peptides is the immunogenic nature of most peptides. In a patient, an immunogenic response to an administered peptide can neutralize the peptide and/or lead to the development of an allergic response in the patient. Other deficiencies of therapeutic glycopeptides include suboptimal potency and rapid clearance rates. The problems inherent in peptide therapeutics are recognized in the art, and various methods of eliminating the problems have been investigated. For example, to provide soluble peptide therapeutics, synthetic polymers have been attached to the peptide backbone.
The attachment of synthetic polymers to the peptide backbone to improve the pharmacokinetic properties of glycoprotein therapeutics is known in the art. An exemplary polymer that has been conjugated to peptides is poly(ethylene glycol) (“PEG”). The use of PEG to derivatize peptide therapeutics has been demonstrated to reduce the immunogenicity of the peptides. For example, U.S. Pat. No. 4,179,337 (Davis et al.) discloses non-immunogenic polypeptides such as enzymes and peptide hormones coupled to polyethylene glycol (PEG) or polypropylene glycol. Between 10 and 100 moles of polymer are used per mole of polypeptide and at least 15% of the physiological activity is maintained. In addition to reduced immunogenicity, the clearance time in circulation is prolonged due to the increased size of the PEG-conjugate of the polypeptides in question.
The principal mode of attachment of PEG, and its derivatives, to peptides is a non-specific bonding through a peptide amino acid residue (see e.g., U.S. Pat. Nos. 4,088,538 4,496,689, 4,414,147, 4,055,635, and PCT WO 87/00056). Another mode of PEG-to-peptide attachment is through the non-specific oxidation of glycosyl residues on a glycopeptide (see e.g., WO 94/05332).
In many chemical PEGylation methods, poly(ethyleneglycol) is added in a random, non-specific manner to reactive residues on a peptide backbone. The random addition of PEG molecules has its inherent disadvantages, including, e.g. the lack of homogeneity in the final product and potential for reduction in the biological or enzymatic activity of the peptide. Therefore, a derivitization strategy that results in the formation of a specifically labeled, readily characterizable, essentially homogeneous product is far superior in the context of therapeutic peptide production. Such methods have been developed.
Specifically labeled, homogeneous peptide therapeutics can be produced in vitro through the action of enzymes. Unlike the typical non-specific methods for attaching a synthetic polymer or other label to a peptide, enzyme-based syntheses have the advantages of regioselectivity and stereoselectivity. Two principal classes of enzymes that can be employed in the synthesis of labeled peptides are glycosyltransferases (e.g., sialyltransferases, oligosaccharyltransferases, N-acetylglucosaminyltransferases) and glycosidases. These enzymes can be used for the specific attachment of sugars which can be subsequently modified to comprise a therapeutic moiety. Alternatively, glycosyltransferases and modified glycosidases can be used to directly transfer modified sugars to a peptide backbone (see e.g., U.S. Pat. No. 6,399,336, and U.S. Patent Application Publications 20030040037, 20040132640, 20040137557, 20040126838, and 20040142856). Methods combining both chemical and enzymatic synthetic elements are also known (see e.g., Yamamoto et al. Carbohydr. Res. 305: 415-422 (1998) and U.S. Patent Application Publication 20040137557).
In response to the need for improved therapeutic hGH, the present invention provides a glycopegylated hGH that is therapeutically active and which has pharmacokinetic parameters and properties that are improved relative to an identical, or closely analogous, hGH peptide that is not glycopegylated. Furthermore, the present invention provides cost-effective methods by which improved hGH peptides can be produced on an industrial scale.
Glycosyl residues have also been modified to bear ketone groups. For example, Mahal and co-workers (Science 276: 1125 (1997)) have prepared N-levulinoyl mannosamine (“ManLev”), which has a ketone functionality at the position normally occupied by the acetyl group in the natural substrate. Cells were treated with the ManLev, thereby incorporating a ketone group onto the cell surface. See, also Saxon et al., Science 287: 2007 (2000); Hang et al., J. Am. Chem. Soc. 123: 1242 (2001); Yarema et al., J. Biol. Chem. 273: 31168 (1998); and Charter et al., Glycobiology 10: 1049 (2000).
Carbohydrates are attached to glycopeptides in several ways of which N-linked to asparagine and mucin-type O-linked to serine and threonine are the most relevant for recombinant glycoprotein therapeutics. A determining factor for initiation of glycosylation of a protein is the primary sequence context, although clearly other factors including protein region and conformation have their roles. N-linked glycosylation occurs at the consensus sequence NXS/T, where X can be any amino acid but proline.
As previously mentioned, rapid in vivo degradation and clearance rate are other well-known problems that interfere with the optimal desired physiological effects of administered polypeptides. Soon after injection, polypeptides such as human growth hormone are readily proteolyzed by numerous proteases in the blood and lymphatic system. These proteases cleave the human growth hormone in both the amino internal and carboxy terminal regions, thereby fragmenting the protein and reducing the growth effects of hGH. The proteolysis also facilitates clearance of the degraded protein, dramatically reducing the residence time in the body after injection. In light of the above, there is a need for polypeptides with protease resistance and method of producing such polypeptides.
The methods discussed above do not provide access to industrially relevant quantities of modified peptides that substantially retain the pharmacological activity of their unmodified analogues and possess protease resistance.
The present invention answers these needs by providing hGH mutants that contain newly introduced O-linked glycosylation sites, providing flexibility in glycosylation and/or glycoconjugation, e.g., glycoPEGylation of these recombinant hGH mutants. The O-glycosylation mutants optionally further include one or more proteolysis resistant mutation or sites for chemical PEGylation of region(s) most susceptible to proteases. Moreover, the invention provides an industrially practical method for the modification of N- or O-linked mutant hGH peptides with modifying groups such as water-soluble polymers, therapeutic moieties, biomolecules, and the like. Of particular interest are methods in which the modified mutant hGH has improved properties, which enhance its use as a therapeutic or diagnostic agent.